Note: Descriptions are shown in the official language in which they were submitted.
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PREPARATION AND SELECTION OF DONOR CELLS
FOR NUCLEAR TRANSPLANTATION
BACKGROUND OF THE INVENTION
A. Cell Synchrony
S An important tool for cell cycle analysis is the ability to place cells in
the same
phase of cell cycle (e.g., S, M, G' or GZ). Cell synchronization has been
performed for
years and can be performed with or without the aid of chemicals. One of the
best
methods of synchronization uses the fact that spherical, mitotic (M) phase
cells adhere
less firmly to glass surfaces than do interphase cells (e.g., interphase cells
are those cells
in S, G~ or GZ). Therefore, by shaking the cell cultures one can isolate large
numbers of
uncontaminated M phase cells (see JAMES D. WATSON ET AL., MOLECULAR BIOLOGY OF
TIIE GENE 971 (4'h ed., 1987). The non-chemical technique of "shake-off '
works well
with Chinese hamster ovary (CHO) cells and some sublines of HeLa (R. IAN FRESI-
INEY,
CULTURE OF ANIMAL CELLS: A MANUAL OF BASIC TECHNIQUES 384-385 (3rd ed. 1994);
and Zwanenburg, Mutat. Res. 120: 151-9 (1983)). Success comparable to that
observed
with CHOs has been achieved in synchronizing diploid human fibroblasts using
mechanical shake-off (Tobey et al., Exp. Cell Res. 179: 400-16 (1988)). Shake-
off has
also been used to synchronize embryonic quail skeletal myoblasts (Devlin et
al., Dev.
Biol. 95: 175-92 (1983)) and HeLa cells (Wheatley et al., Cytobios. SS: 191-
204 (1988)).
Another mechanical means of synchronizing cells in G~ is to use centrifugal
elutriation,
which can cause the cells temporarily arrest into (Zickert et al., Exp. Cell.
Res. 207:115-
21 ( 1993)).
Cell synchronization can also be achieved by using a combination of mechanical
shake-off and chemicals (e.g., aphidicolin) (Graves et al., Anal. Biochem.
248: 251-7
(1997)). However, use of drugs (e.g., aphidicolin or hydroxyurea) have toxic
side effects
on CHOs, whereas shake-off does not (Fox et al., Cytometry 8: 3 15-20 (1987)).
Drugs
can also be used alone to synchronize cells. G~ and/or Go arresting drugs
include
dexamethasone (Goya et al., Mol. Endocrinol. 7: 1121-32 (1993)), as well as
other
glucocorticoids (Sanchez et al., Cell Growth Differ. 4: 2 1 S-25 (1993)), or
bidentate 3-
hydroxypyridin-4-one (HPO) and hexdentate desfernoxamine (DFO) (Hoyes et al.,
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Cancer Res. 52: 4591-9 (1992)). Other G1-specific cell cycle synchronizing
agents are
discussed in Gadbois et al., Proc. Nat'1 Acad. Sci. USA 89: 8626-8630 (1992).
Temperature has also been employed to mediate the cell cycle of a cell. Cold-
shock synchronizes immature granulocytic cells from peripheral blood or bone
marrow
(Boucher et al., Hum. Genet. 54: 207-11(1980)). Human diploid fibroblasts are
arrested
in G, by switching the cells to low temperature, such as 30°C. (Enninga
et al., Mutat.
Res. 130: 343-52 (1984)). Temperature was used to stop cell cycle in G,, S,
late S and
GZ+M phases after the CHO cells were synchronized using the mechanical shake-
off
procedure (Schneiderman et al., Radiat. Res. 116: 283-91 (1988)).
The cell cycle stage of donor cell nuclei critically affects the chromatin
structure
and development of nuclear transplant embryos. Synchronization of the donor
nucleus in
the G, phase is an important factor for successful development of nuclear
transplant
embryos (Cheong et al., Biol. Reprod. 48: 958-63 (1993)). Specifically, late S
chromatin
influences chromosome constitution in embryos and may account for the reduced
development of nuclear transplant embryos when late S phase donor nuclei are
used
(Collas et al., Biol. Reprod. 46: 501-11 (1992)). Cell cycle influences the
use of a donor
nucleus on chromatin structure and development of mouse embryonic nuclei
transplanted
into enucleated oocytes. Cell-cycle synchronization has also been shown to
play an
important role in the use of porcine ectodermal cell donor nuclei in nuclear
reprogramming of the nuclei material after the donor is fused to an enucleated
metaphase-
Il oocyte (Ouhibi et al., Mol. Reprod. Dev. 44: 533-9 (1996)). However, cell
synchronization, for purposes of nuclear transplantation of somatic cell
nuclei, has not
used mitotic cell shake-off in combination with doublet cell selection.
Moreover, the
shake-off and doublet selection of somatic cells has not been used in
combination with
other methods of cell cycle synchronization (e.g., Gl phase arresting agents
or methods)
for the purpose of preparing somatic cell nuclei for transplantation.
B. Preparing Somatic Cells for Nuclear Transplantation or Nuclear Transfer
In 1996, the first successful transfer of a nucleus from an adult mammary
gland
cell into an enucleated oocyte was reported (Campbell et al., Nature 380: 64-6
(1996)).
This success was followed by the production of rhesus monkeys by nuclear
transfer of
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embryonic cells. Nuclear transfer involves preparing a cytoplast as a
recipient cell. In
most cases, the cytoplast is derived from a mature metaphase II oocyte from
which the
chromosomes have been removed. A donor cell nucleus is then placed between the
zona
and the cytoplast; fusion, as well as cytoplast activation, is initiated by
electrical
stimulation. Successful reprogramming of the donor cell nucleus by the
cytoplast is
critical, and is a step which may be influenced by cell cycle. See Wolf et
al., Biol.
Reprod. 60: 199-204 (1999). A number of pregnancies have been established
using fetal
cells as the source of donor nuclei. However, the use of cell lines to create
transgenic
animals permits large clone sizes and the genetic manipulation of the cells in
vitro before
nuclear transfer. Id. The mechanisms regulating early embryonic development
may be
conserved among mammalian species, such that, for example, bovine oocyte
cytoplasm
can support the introduced differentiated donor nucleus regardless of
chromosome
number, species or age of the donor fibroblast (Dominko et al., Biol. Reprod.
60: 1496-
1502 ( 1999)).
Actively dividing fetal fibroblasts can be used as nuclear donors according to
the
procedure described in Cibelli et al., Science 280: 1256-9 (1998). Additional
methods of
preparing recipient oocytes for nuclear transfer of donor differentiated
nuclei are as
described in International PCT Application Nos. 99105266; 99/01164; 99/01163;
98/3916;
98/30683; 97/41209; 97/07668; and U.S. Patent No. 5,843,754. Typically the
transplanted nuclei are from cultured embryonic stem (ES), embryonic germ (EG)
cells or
other embryonic cells. See International PCT Applications Nos. 95/17500 and
95/10599;
Canadian Patent No. 2,092,258; Great Britain Patent No. 2,265,909; and U.S.
Patent Nos.
5,453,366; 5,057,420; 4,994,384; and 4,664,097. Inner cell mass (ICM) cells
can also be
used as nuclear donors (Sims et al., Proc. Natl Acad. Sci. USA 90: 6143-6147
(1990); and
Keefer et al., Biol. Reprod. S0: 935-939 (1994)).
C. Transgenic Animals and Production Thereof
Pronuclear Microinjection. Various methods have been utilized in an attempt to
genetically modify animals so as to introduce superior qualities including
pronuclear
microinjection. One of the limitations of pronuclear microinjection is that
the gene
insertion site is random. This typically results in variation of expression
levels, and
several transgenic lines must be produced to obtain one line with an
appropriate level of
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expression. Because integration is random, it is advantageous that lines of
transgenic
animals are started from one founder animal to avoid difficulties in
monitoring zygosity
and potential difficulties that might occur with interactions among multiple
insertion sites
(Cundiff et al., J. Animal Sci. 71: 20-25 (1993)). Even without concern for
inbreeding, it
would take about 6.5 years before reproduction could be tested in homozygous
animals
(Seidel, J. Animal Sci. 71: 26-33 (1993)).
A second limitation of the pronuclear microinjection procedure is its
efficiency.
Only 0.34 to 2.63% of the gene-injected embryos develop into transgenic
animals, and a
fraction of these appropriately express the gene (Purcel et al., J. Animal
Sci. 71:10-19
(1993)). This inefficiency results in a high cost of producing transgenic
animals because
of the large number of recipients required. Thus, the ability to clone, or to
make
numerous identical genetic copies, of an animal comprising a desired genetic
modification would be advantageous.
Embryonic Stem Cells. Another system for producing transgenic animals has
been developed that uses embryonic stem (ES) cells. In mice, ES cells have
enabled
researchers to select for transgenic cells and perform gene targeting. This
method allows
more genetic engineering than is possible with other transgenic techniques.
For example,
ES cells are relatively easy to grow as colonies in vitro, can be transfected
by standard
procedures, and the transgenic cells clonally selected by antibiotic
resistance (T.
Doetschman, "Gene transfer in embryonic stem cells." IN TRANSGENIC ANIMAL
TECHNOLOGY: A LABORATORY HANDBOOK 11 S-146 (C. Pinkert, ed., Academic Press,
Inc., New York 1994)). Furthermore, the efficiency of this process is such
that sufficient
transgenic colonies (hundreds to thousands) can be produced to allow a second
selection
for homologous recombinants. Id. ES cells can then be combined with a normal
host
embryo and, because they retain their potency, and can develop into all the
tissues in the
resulting chimeric animal, including the germ cells. Therefore, transgenic
modification is
transmissible to subsequent generations.
Methods for deriving embryonic stem (ES) cell lines in vitro from early
preimplantation mouse embryos are well known (Evans et al., Nature 29: 154-156
(1981); Martin, Proc. Natl. Acad. Sci. USA 78: 7634-7638 (1981)). ES cells can
be
passaged in an undifferentiated state, provided that a feeder layer of
fibroblast cells
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(Evans et al., 1981) or a differentiation inhibiting source (Smith et al.,
Dev. Biol. 121:1-9
( 1987)) is present.
In view of their ability to transfer their genome to the next generation, ES
cells
have potential utility for germline manipulation of livestock animals. Some
research
groups have reported the isolation of purportedly pluripotent embryonic cell
lines. For
example, Notarianni et al., J. Reprod. Fert. Suppl. 43: 55-260 (1991) reported
the
establishment of stable, pluripotent cell lines from pig and sheep
blastocysts, which
exhibit some morphological and growth characteristics similar to that of cells
in primary
cultures of inner cell masses (ICMs) isolated immunosurgically from sheep
blastocysts.
Also, Notarianni et al., J. Reprod. Fert. Suppl. 41: 51-56 (1990) disclosed
maintenance
and differentiation in culture of putative pluripotent embryonic cell lines
from pig
blastocysts. Gerfen et al., Anim. Biotech. 6:1-14 (1995) disclosed the
isolation of
embryonic cell lines from porcine blastocysts. These cells are stably
maintained without
mouse embryonic fibroblast feeder layers and reportedly differentiate into
several
different cell types during culture.
Further, Saito et al., Roux's Arch. Dev. Bid. 201: 134-141 (1992) reported
cultured, bovine embryonic stem cell-like cell lines, which survived three
passages, but
were lost after the fourth passage. Handyside et al., Roux's Arch. Dev. Biol.
196:185-190
( 1987) disclosed culturing immunosurgically isolated inner cell masses (ICMs)
of sheep
embryos under conditions that allow for the isolation of mouse ES cell lines
derived from
mouse ICMs.
Chemy et al., Theriogenology 41:175 (1994) reported purportedly pluripotent
bovine primordial germ cell-derived cell lines maintained in long-term
culture. These
cells, after approximately seven days in culture, produced ES-like colonies,
which stained
positive for alkaline phosphatase (AP), exhibited the ability to form embryoid
bodies, and
spontaneously differentiated into at least two different cell types.
Campbell et al., ( 1996) reported the production of live lambs following
nuclear
transfer of cultured embryonic disc (ED) cells from day nine ovine embryos
cultured
under conditions which promote the isolation of ES cell lines in the mouse.
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Van Stekelenburg-Hamers et al., Mol. Reprod. Dev. 40: 444-454 (1995) reported
the isolation and characterization of purportedly permanent cell lines from
ICMs of
bovine blastocysts. The authors isolated and cultured ICMs from eight or nine
day
bovine blastocysts under different conditions to determine which feeder cells
and culture
media are most efficient in supporting the attachment and outgrowth of bovine
ICM cells.
Purportedly, animal stem cells have been isolated, selected and propagated for
use
in obtaining transgenic animals (see Evans et al., WO 90/03432; Smith et al.,
WO
94/24274; and Wheeler et al., WO 94/26884). Evans et al. also reported the
derivation of
purportedly pluripotent ES cells from porcine and bovine species, which
assertedly are
useful for the production of transgenic animals.
ES cells from a transgenic embryo could be used in nuclear transplantation.
The
use of ungulate ICM cells for nuclear transplantation also has been reported.
In the case
of livestock animals, e.g., ungulates, nuclei from similar preimplantation
livestock
embryos support the development of enucleated oocytes to term (Keefer et al.,
1994;
Smith et al., Biol. Reprod. 40:1027-1035 (1989)). In contrast, nuclei from
mouse
embryos do not support development of enucleated oocytes beyond the eight-cell
stage
after transfer (Cheong et al., Biol. Reprod. 48: 958 (1993)). Therefore, ES
cells from
livestock animals are highly desirable, because they may provide a potential
source of
totipotent donor nuclei, genetically manipulated or otherwise, for nuclear
transfer
procedures.
Use of ICM Cells. Collas et al., Mol. Reprod. Dev. 38: 264-267 (1994)
disclosed
nuclear transplantation of bovine ICMs by microinjection of the lysed donor
cells into
enucleated mature oocytes. Culturing of embryos in vitro for seven days
produced fifteen
blastocysts which, upon transfer into bovine recipients, resulted in four
pregnancies and
two births. Also, Keefer eta?., Biol. Reprod. S0: 93 5-939 (1994) disclosed
the use of
bovine ICM cells as donor nuclei in nuclear transfer procedures, to produce
blastocysts
which also resulted in several live offspring. Further, Sims et al., Proc.
Natl. Acad. Sci.
USA 90: 6143-6147 (1993) disclosed the production of calves by transfer of
nuclei from
short-term in vitro cultured bovine ICM cells into enucleated mature oocytes.
Therefore, notwithstanding what has previously been reported in the
literature,
there exists a need for improved methods of preparing large numbers of cells
for nuclear
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transfer or transplantation for use in creating transgenic or chimeric
animals. Using cell
cycle-synchronized cells, which represent a rapidly dividing sub-population,
as donor
nuclei will enhance the development of transgenic or chimeric animals,
especially
livestock animals, using nuclear transfer procedures.
SUMMARY AND OBJECTS OF THE INVENTION
It is an object of the present invention to provide a method of preparing
donor
somatic cells for nuclear transfer or nuclear transplantation comprising the
steps of: (A)
synchronizing the cell cycle of donor somatic cells by shaking the cells; (B)
selecting
doublet cells from said shaken somatic cells; and (C) preparing the selected
mitotic
double cells for nuclear transfer. A cooling step can optionally be included
wherein the
selected doublet cells are cooled to below metabolic temperature in order to
lengthen the
G~ phase. Also, optionally, the numbers of cells in G~ phase or the period of
GI can be
enhanced by placing the cells in appropriate media, e.g., media lacking at
least one of the
following: serum, isoleucine, glutamine or phosphate or by the addition of a
G~
synchronizing agent (e.g., aphidicolin or mimosine).
It is a specific object of the invention to obtain cells which are rapidly
dividing
somatic cells, for example (cell cycle completion in 15 hours or less, more
preferably 10
hours or less). Such cells can be obtained using the methods described above.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: Effect of Confluency and Cell Age on Cell Cycle Length. The histogram
describes the difference in cell cycle lengths (measured in hours) of cells at
25%
confluence versus cells at 90% confluency. Cell cycles were observed in cell
populations
obtained from 40 day old fetuses (40D FET), 4 year old cows (4 YRS), 15 year
old cows
(15 YRS) and total cells.
FIG. 2: Effect of Time in Culture and Donor Age on Cell Growth Rate. The cell
growth rate was compared for cells derived from 40 day old fetuses (40D FET),
calves
aged from 0-13 months (0-13 MO) and calves aged 24-72 months (24-72).
Population
doubling (PD) is compared depending on the number of days the cells are in
culture. The
mean PD decreases as the number of days in culture increases.
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FIG. 3: Length of Gl in Fibroblasts Recovered from Culture.
DETAILED DESCRIPTION OF THE INVENTION
For a better understanding of the invention, the following detailed
description
refers to the accompanying drawings and examples, wherein preferred exemplary
embodiments of the present invention are illustrated and described. This
invention relates
to a novel method of obtaining somatic cells as donor nuclei, which provides a
population
of donor nuclei temporally optimized for nuclear transfer or nuclear
transplantation.
A. Definitions
By "synchronized cells" or "synchronizing" is meant a culture of cells or a
method of preparing said cells such that more than 90% of the cells are in G1
phase.
By "confluent cells" is meant cell population densities of about 90% or
greater.
The terms "nuclear transfer" or "nuclear transplantation" refer to a method of
cloning, wherein the donor cell nucleus is transplanted into a cell cytoplast.
The cytoplast
could be from an enucleated oocyte, an enucleated ES cell, an enucleated EG
cell, an
1 S enucleated embryonic cell or an enucleated somatic cell. Nuclear transfer
techniques or
nuclear transplantation techniques are known in the literature (Campbell et
al.,
Theriogenology 43: 181 (1995); Collas et al., (1994); Keefer et al., (1994);
Sims et al.,
(1993); Evans et al., WO 90/03432; Smith et al., WO 94/24274; and Wheeler et
al., WO
94/26884. Also U.S. Patent Nos. 4,994,384 and 5,057,420 describe procedures
for bovine
nuclear transplantation. In the subject application, "nuclear transfer" or
"nuclear
transplantation" or "NT" are used interchangeably.
The terms "nuclear transfer unit" and "NT unit" refer to the product of fusion
between or injection of a somatic cell or cell nucleus and an enucleated
cytoplast (e.g., an
enucleated oocyte), which is sometimes referred to herein as a fused NT unit.
By "somatic cell" is meant any cell of a multicellular organism, preferably an
animal, that does not become a gamete. The preferred "somatic cell" are
adherent cells.
By "adherent cells" are meant cells that when cultured adhere to the surface
of the tissue
culture flask or other such compartment.
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By "animal" is meant to include mammals, e.g. livestock animals (e.g.,
ungulates
such as cattle, buffalo, horses, sheep, pigs and goats), as well as rodents
(e.g., mice,
hamsters, rats and guinea pigs), domesticated animals such as canines, felines
horses,
rabbits and primates. Animals also include endangered or even extinct species
such as
S guar, giant pandas, elephants, a African bongo antelope, Sumatran tiger,
bucardo
mountain goat, cheetah, and ocelot, et seq.
By "doublet cell" is meant to include those cells which are attached by
cytoplasmic bridges. A "cytoplasmic bridge" occurs during the final stages of
cytokinesis, before the daughter cells complete separation.
By "rapidly dividing cell" is meant a cell being grown in a low population
density
(50% population density or less) in media containing serum.
By "G1 synchronizing agent" is meant an agent which enhances the production of
cells in, or arrests a cell in, Gl.
By "chimera" or "chimeric animal" is meant an organism composed of two
genetically distinct types of cells. The chimera can be formed by the fusion
of two early
blastula stage embryos, for example.
By "transgenic animal" is mean an organism that has integrated into its genome
one or more foreign DNA molecules.
B. Cell Cycle Synchronization
Somatic cell synchronization will be performed using mitotic shake-off wherein
cells are shaken by slapping tissue culture flasks to knock-off mitotic cells
from the flask
wall. In brief 0.5X106 cells are plated 24 hours prior to shake-off. Shake-off
is carried
out typically by placing the flask or other tissue culture dish on a vortexer
or other
shaking apparatus are for about 30 to about 60 seconds. Media containing the
cells which
are shaken off is removed and centrifuged. The pelleted cells are resuspended
in 250 "u1
of medium. Doublet cells are separated from non-doublet cells obtained at the
shake-off
step by visual inspection. Doublet cells can also be isolated, e.g., by
centrifugation using
a gradient to separate doublet cells from non-doublet cells.
These cells can immediately be used for nuclei removal for nuclear
transplantation
or nuclear transfer. Alternatively, the cells can be cooled to below metabolic
temperature
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(e.g., below 37°C., more preferably 4-20°C., and most preferred
at 4°C.) to maintain the
period that they are in G~. The cells can also be kept in G~ phase using other
means, such
as serum deprivation or depletion of isoleucine, glutamine or phosphate from
the media
after doublet selection. Drugs, such as colchicine, blocks cells in M phase
(JAMES D.
WATSON ETAL., MOLECULAR BIOLOGY OF THE GENE 973 (4th ed., 1987). Other drugs
can
block cells in Gl phase, such as mimosine (Krude, Exp. Cell. Res. 247:148-
59(1999)),
glucocorticoids (Sanchez et al., Cell Growth Differ. 4: 2 15-25 (1993))
aphidicolin and
certain kinase inhibitors (e.g., KT5720, KT5823, KT5926 and K5256 described in
Gadbois et al., 1992). Other drugs block cells at the Gl-S border, including
bidentate 3-
hydroxypyridin-4-one iron chelators and hexadentate desfernxoxamine (Hoyes et
al.,
Cancer Res. 52: 459 1-9 (1992)). These drugs can be added to the media of the
selected
doublet cells to lengthen the period of the G~ phase.
C. Nuclear Transplantation and Development of Transgenic Animals Using Somatic
Cells
The use of adult cells and fetal fibroblast cells from a sheep have been used
as
nuclear transfer donors to produce a cloned sheep offspring (Wilmut et al.,
Nature 385:
810-813 (1997)). However, in that study, it was emphasized that the use of a
serum
starved, nucleus donor cell in the quiescent state was important for success
of the Wihnut
cloning method. No such requirement for serum starvation or quiescence for
maintaining
the cells in Go exists for the present invention. On the contrary, cloning is
achieved using
differentiated mammalian cells proceeding through cell cycle, i.e., cells in
G~, GZ or M or
S phases.
Thus, in one aspect, the present invention provides an improved method for
cloning an animal. In general, the animal will be p Lourenco roduced by a
nuclear
transfer process comprising the following steps:
(i) obtaining desired somatic cells by the methods described herein, which
may be serum or non-serum starved, to be used as a source of donor nuclei;
(ii) obtaining oocytes from an animal, e.g., bovine;
(iii) enucleating said oocytes;
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(iv) transfernng the desired somatic cell or cell nucleus into the enucleated
oocyte, e.g., by fusion or injection, to form an NT unit;
(v) (v) activating the NT unit to yield an activated NT unit; and
(vi) (vi) transferring said activated NT unit to a host animal such that the
NT
unit develops into a fetus.
Optionally, the activated NT unit is cultured beyond the 2-cell developmental
stage prior to transfer to the host animal.
The present invention also includes a method of cloning a genetically
engineered
or transgenic animal, by which a desired DNA sequence is inserted, removed or
modified
in the serum or non-serum starved differentiated animal cell or cell nucleus
prior to
insertion of the differentiated animal cell (e.g., somatic cell) or cell
nucleus into an
oocyte, which is enucleated before or after nuclear transfer.
In addition to the uses described above, the genetically engineered or
transgenic
animals according to the invention can be used to produce a desired protein,
such as a
pharmacologically important protein, e.g., human serum albumin. That desired
protein
can then be isolated from milk or other fluids or tissues of the transgenic
animal.
Alternatively, the exogenous DNA sequence may confer an agriculturally useful
trait to
the transgenic animal, such as disease resistance, decreased body fat,
increased lean meat
product, improved feed conversion, or altered sex ratios in progeny.
The stage of oocyte maturation at enucleation and nuclear transfer has been
reported to be significant to the success of NT methods (Prather et al.,
Differentiation 48:
1-8 ( 1991 )). In general, successful mammalian embryo cloning practices use a
metaphase
II stage oocyte as the recipient oocyte, because at this stage it is believed
that the oocyte
can reprogram the nucleus by causing disassembly of the nucleus and
condensation of the
chromatin. Activation of the NT unit is then induced. In domestic animals, the
oocyte
activation period generally ranges from about 16-52 hours, preferably about 20-
45 hours
post-aspiration.
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Methods for isolating of oocytes are well known in the art. Essentially, this
comprises isolating oocytes from the ovaries or reproductive tract of an
animal, e.g., a
bovine. A readily available source of bovine oocytes is from slaughterhouse
materials.
For the successful use of techniques such as genetic engineering, nuclear
transfer
and cloning, oocytes must generally be matured in vitro before these cells may
be used as
recipient cells for nuclear transfer, and before they can be fertilized by the
sperm cell to
develop into an embryo. This process generally requires collecting immature
(prophase I)
oocytes from mammalian ovaries, e.g., bovine ovaries obtained from a
slaughterhouse,
and maturing the oocytes in a maturation medium prior to fertilization or
enucleation until
the oocyte attains the metaphase II stage, which in the case of bovine oocytes
generally
occurs about 18-24 hours post-aspiration. For proposes of the present
invention, this
period of time is known as the "maturation period." As used herein for
calculating time
periods, "aspiration" refers to aspiration of the immature oocyte from ovarian
follicles.
Additionally, metaphase II stage oocytes, which have been matured in vivo,
have
been successfully used in nuclear transfer techniques. Essentially, mature,
cow
metaphase II oocytes can be collected surgically from either non-superovulated
or
superovulated cows or heifers from about 20 to about 30 hours past the onset
of estrus or
past the injection of human chorionic gonadotropin (hCG) or similar hormone.
The stage of maturation of the oocyte at enucleation and nuclear transfer has
been
reported to be significant to the success of NT methods. (See, Prather et al.,
Differentiation 48: 1-8 (1991)). In general, successful mammalian embryo
cloning
practices use the metaphase II stage oocyte as the recipient oocyte, because
at this stage it
is believed that the oocyte can be or is sufficiently "activated" to treat the
introduced
nucleus as it does a fertilizing sperm. In domestic animals, and especially
cattle, the
oocyte activation period generally ranges from about 16-52 hours, preferably
about 2 8-
42 hours post-aspiration.
For example, immature oocytes may be washed in HEPES buffered hamster
embryo culture medium (HECM), as described in Seshagine et al., Biol. Reprod.,
40:544-
606 (1989), and then placed into drops of maturation medium consisting of 50
~C1 of
tissue culture medium (TCM) 199 containing 10% fetal calf serum, which further
contains appropriate gonadotropins such as luteinizing hormone (LH) and
follicle
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stimulating hormone (FSH), and estradiol under a layer of lightweight paraffin
or silicon
at 39°C.
After a fixed time maturation period, which ranges from about 10 to about 40
hours, and preferably about 16-18 hours, the oocytes will be enucleated. Prior
to
enucleation the oocytes will preferably be removed and placed in HECM
containing 1
mg/ml of hyaluronidase prior to removal of cumulus cells. This may be effected
by either
repeated pipetting through very fine bore pipettes or by vortexing briefly.
The stripped
oocytes are then screened for polar bodies, and the selected metaphase II
oocytes, as
determined by the presence of polar bodies, are then used for nuclear
transfer.
Method of Enucleating Cells. Enucleation may be effected by known methods,
such as described in U.S. Patent No. 4,994,384, which is incorporated by
reference
herein. For example, metaphase II oocytes are either placed in HECM,
optionally
containing 7.5 ,ug/ml cytochalasin B, for immediate enucleation, or may be
placed in a
suitable medium, for example an embryo culture medium, such as CRl as (CR1
media is
described in U.S. Patent No. 5,096,822. CRlaa is supplemented with amino
acids), plus
10% estrus cow serum, and then enucleated later, preferably not more than 24
hours later,
and more preferably 16-18 hours later.
Enucleation may be accomplished microsurgically using a micropipette to remove
the polar body and the adjacent cytoplasm. The oocytes may then be screened to
identify
those of which have been successfully enucleated. This screening may be
effected by
staining the oocytes with 1 ,ug/ml 33342 Hoechst dye in HECM, and then viewing
the
oocytes under ultraviolet irradiation for less than 10 seconds. Oocytes
successfully
enucleated can then be placed in a suitable culture medium, e.g., CRlaa
supplemented
with 10% serum.
In the present invention, the recipient oocytes will preferably be enucleated
at a
time ranging from about 10 hours to about 40 hours after the initiation of in
vitro
maturation, more preferably from about 16 hours to about 24 hours after
initiation of in
vitro maturation, and most preferably about 16-18 hours after initiation of in
vitro
maturation.
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A single mammalian somatic cell of the same species or different species will
then
be transferred into the perivitelline space of the oocyte used to produce the
NT unit. Very
recently, it was reported that to transfer of guar cells into an enucleated
bovine oocyte
resulted in a viable embryo (Scientific American Lonza et al., October 2000).
The
mammalian cell and the oocyte will be used to produce NT units according to
methods
known in the art. For example, the cells may be fused by electrofusion.
Electrofusion is
accomplished by providing a pulse of electricity sufficient to cause a
transient breakdown
of the plasma membrane. This breakdown of the plasma membrane is very short-
lived,
because the membrane reforms rapidly. Thus, if two adjacent membranes are
induced to
breakdown and upon reformation the lipid bilayers intermingle, small channels
will open
between the two cells. Due to the thermodynamic instability of such a small
opening, it
enlarges until the two cells become one. Reference is made to U.S. Patent
4,997,384 by
Prather et al., (incorporated herein by reference in its entirety) for a
further discussion of
this process. A variety of electrofusion media can be used including, e.g.,
sucrose,
mannitol, sorbitol and phosphate buffered solution. Fusion can also be
accomplished
using Senclai virus as a fusogenic agent (Graham, Wistar Inst. Symp. Monogr.
9:19
( 1969)).
Also, in some cases (e.g., with small donor nuclei) it may be preferable to
inject
the nucleus directly into the oocyte rather than using electroporation fusion.
Such
techniques are disclosed in Collas et al., Mol. Reprod. Dev., 38: 264-267
(1994),
incorporated by reference in its entirety herein.
Preferably, the somatic or germ cell and oocyte are electrofused in a 500 p.m
chamber by application of an electrical pulse of about 90-120 V for about 15
,usec, about
24 hours after initiation of oocyte maturation. After fusion, the resultant
fused NT units
are then placed in a suitable medium until activation, e.g., CRlaa medium.
Typically
activation will be effected shortly thereafter, typically less than 24 hours
later, and
preferably about 4-9 hours later.
The NT unit may be activated by known methods. Such methods include, e.g.,
culturing the NT unit at sub-physiological temperature, in essence by applying
a cold, or
actually cool temperature shock to the NT unit. This may be most conveniently
done by
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culturing the NT unit at room temperature, which is cold relative to the
physiological
temperature conditions to which embryos are normally exposed.
Alternatively, activation may be achieved by application of known activation
agents. For example, penetration of oocytes by sperm during fertilization has
been shown
to activate prefusion oocytes to yield greater numbers of viable pregnancies
and multiple
genetically identical calves after nuclear transfer. Also, treatments such as
electrical and
chemical shock may be used to activate NT embryos after fusion. Suitable
oocyte
activation methods are the subject of U.S. Patent No. 5,496,720, to Susko-
Parnsh et al.,
herein incorporated by reference in its entirety.
Additionally, activation may be affected by simultaneously or sequentially:
(i) increasing levels of divalent cations in the oocyte, and
(ii) reducing phosphorylation of cellular proteins in the oocyte.
This will generally be effected by introducing divalent cations into the
oocyte cytoplasm,
e.g., magnesium, strontium, barium or calcium, e.g., m the form of an
ionophore. Other
methods of increasing divalent cation levels include the use of electric
shock, treatment
with ethanol and treatment with caged chelators.
Phosphorylation may be reduced by known methods, e.g., by the addition of
kinase inhibitors, (e.g., serine-threonine kinase inhibitors, such as 6-
dimethyl-
aminopurine, staurosporine, 2-aminopurine, and sphingosine).
Alternatively, phosphorylation of cellular proteins may be inhibited by
introduction of a phosphatase into the oocyte, e.g., phosphatase 2A and
phosphatase 2B.
In one embodiment, NT activation is effected by briefly exposing the fused NT
unit to a TL-HEPES medium containing 5 ,uM ionomycin and 1 mg/ml BSA, followed
by
washing in TL-HEPES containing 30 mg/ml BSA within about 24 hours after
fusion, and
preferably about 4 to about 9 hours after fusion.
The activated NT units may then be cultured in a suitable in vitro culture
medium
until the generation of cultured inner cell mass (CICM) cells and cell
colonies. Culture
media suitable for culturing and maturation of embryos are well known in the
art.
Examples of known media, which may be used for bovine embryo culture and
CA 02387488 2002-04-10
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maintenance, include Ham's F-10 + 10% fetal calf serum (FCS), Tissue Culture
Medium-
199 (TCM- 199) supplemented with 10% fetal calf serum, Tyrodes-Albumin-Lactate-
Pyruvate (TALP), Dulbecco's Phosphate Buffered Saline (PBS), Eagle's and
Whitten's
media. A common media used for the collection and maturation of oocytes is TCM-
199,
supplemented with 1 to 20% FCS, newborn serum, estrual cow serum, lamb serum
or
steer serum. A preferred maintenance medium includes TCM-199 with Earl salts,
10%
fetal calf serum, 0.2 mM Na pyruvate and SO,ug/ml gentamicin sulphate. Any of
the
above may also involve co-culture with a variety of cell types, such as
granulosa cells,
oviduct cells, BRL cells and uterine cells and STO cells.
Another maintenance medium is described in U.S. Patent 5,096,822 to
Rosenkrans, which is incorporated herein by reference. This embryo medium,
named
CR1, contains the nutritional substances necessary to support an embryo.
For example, the activated NT units may be transferred to CRlaa culture medium
containing 2.0 mM DMAP (Sigma) and cultured under ambient conditions, e.g.,
about
38.5°C., 5% COZ for a suitable time, e.g., about 4 to about S hours.
Afterward, the cultured NT unit or units are preferably washed and then placed
in
a suitable media, e.g., CRlaa medium containing 10% FCS and 6 mg/ml contained
20 in
well plates, which preferably contain a suitable confluent feeder layer.
Suitable feeder
layers include, by way of example, fibroblasts and epithelial cells, e.g.,
fibroblasts and
uterine epithelial cells derived from ungulates, chicken fibroblasts, marine
(e.g., mouse or
rat) fibroblasts, STO and SI-m220 feeder cell lines, and BRL cells.
The methods for embryo transfer and recipient animal management in the present
invention are standard procedures used in the embryo transfer industry.
Synchronous
transfers are important for success of the present invention, i.e., the stage
of the NT
embryo is in synchrony with the estrus cycle of the recipient female. This
advantage and
how to maintain recipients are reviewed in Seidel, "Critical review of embryo
transfer
procedures with cattle" IN FERTILIZATION AND EMBRYONIC DEVELOPMENT IN VITRO (L
Mastroianni, Jr. et al., eds., Plenum Press, New York, NY, 1981), the contents
of which
are hereby incorporated by reference.
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The present invention can also be used to clone genetically engineered or
transgenic animals. As explained above, the present invention is advantageous
in that
transgenic procedures can be simplified by working with a somatic cell source
that can be
clonally propagated. In particular, the somatic cells used for donor nuclei,
which may or
may not be serum-starved, have a desired DNA sequence inserted, removed or
modified.
Those genetically altered, somatic cells are then used for nuclear
transplantation with
enucleated oocytes.
Any known method for inserting, deleting or modifying a desired DNA sequence
from a mammalian cell may be used for altering the somatic cell to be used as
the nuclear
donor. These procedures may remove all or part of a DNA sequence, and the DNA
sequence may be heterologous. Included is the technique of homologous
recombination,
which allows the insertion, deletion or modification of a DNA sequence or
sequences at a
specific site or sites in the cell genome. A preferred method is the
positive/negative
selection method patented by Capecchi (U.5. Patent No. 5,631,153, 5,627,059,
and
5,847,982) or vectors reported in U.S. Patent 6,110,735, 5,948,653, 5,925,577,
5,830,698,
5,776,777, 5,763,290, 5,574,205, and 5,527,644, all of which are incorporated
by
reference in their entirety.
The present invention can thus be used to provide adult animals, such as cows,
with desired genotypes. Multiplication of adult animals with proven genetic
superiority
or other desirable traits is particularly useful, including transgenic or
genetically
engineered animals, and chimeric animals. Thus, the present invention will
allow
production of single sex offspring, and production of animals having improved
meat
production, reproductive traits and disease resistance. Furthermore, cell and
tissues from
the NT fetus, including transgenic and/or chimeric fetuses, can be used in
cell, tissue and
organ transplantation for the treatment of numerous diseases, as described
below, in
connection with the use of CICM cells. Hence, transgenic animals have uses
including
models for diseases, xenotransplantation of cells and organs, and production
of
pharmaceutical proteins.
For production of CICM cells and cell lines, the activated NT units are
cultured
under conditions which promote cell division without differentiation to
provide for
cultured NT units. After cultured NT units of the desired size are obtained,
the cells are
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mechanically removed from the zona pellucida and are then used. This is
preferably
effected by taking the clump of cells which comprise the cultured NT unit,
which
typically will contain at least about SO cells, washing such cells, and
plating the cells onto
a feeder layer, e.g., irradiated fibroblast cells. Typically, the cells used
to obtain the stem
cells or cell colonies will be obtained from the inner most portion of the
cultured NT unit
which is preferably at least 50 cells in size. However, cultured NT units of
smaller or
greater cell numbers as well as cells from other portions of the cultured NT
unit may also
be used to obtain ES cells and cell colonies. The cells are maintained on the
feeder layer
in a suitable growth medium, e.g., alpha MEM supplemented with 10% FCS and 0.1
mM
13-mercaptoethanol (Sigma) and L-glutamine. The growth medium is changed as
often as
necessary to optimize growth, e.g., about every 2-3 days.
This culturing process results in the formation of CICM cells or cell lines.
One
skilled in the art can vary the culturing conditions as desired to optimize
growth of the
particular CICM cells. Also, for example, genetically engineered or transgenic
cow
CICM cells may be produced according to the present invention. That is, the
methods
described above can be used to produce NT units in which a desired DNA
sequence or
sequences have been introduced, or from which all or part of an endogenous DNA
sequence or sequences have been removed or modified. Those genetically
engineered or
transgenic NT units can then be used to produce genetically engineered or
transgenic
CICM cells.
The resultant CICM cells and cell lines have numerous therapeutic and
diagnostic
applications. Most especially, such CICM cells may be used for cell
transplantation
therapies.
In this regard, it is known that mouse embryonic stem (ES) cells are capable
of
differentiating into almost any cell type, e.g., hematopoietic stem cells.
Therefore, the
CICM cells produced according to the invention should possess similar
differentiation
capacity. The CICM cells according to the invention will be induced to
differentiate to
obtain the desired cell types according to known methods. For example, the
subj ect cow
CICM cells may be induced to differentiate into hematopoietic stem cells,
neural cells,
muscle cells, cardiac muscle cells, liver cells, cartilage cells, epithelial
cells, urinary tract
cells, neural cells, etc., by culturing such cells in differentiation medium
and under
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conditions which provide for cell differentiation. Medium and methods which
result in
the differentiation of CICM cells are known in the art as are suitable
culturing conditions.
For example, Palacios, et al., Proc. Natl. Acad. Sci. USA 92: 7530-7 (1995)
teaches the production of hematopoietic stem cells from an embryonic cell line
by
subjecting stem cells to an induction procedure comprising initially culturing
aggregates
of such cells in a suspension culture medium lacking retinoic acid followed by
culturing
in the same medium containing retinoic acid, followed by transferral of cell
aggregates to
a substrate which provides for cell attachment.
Moreover, Pedersen, J. Reprod. Fertil. Dev. 6: 543-552 (1994), a review
article,
references numerous articles disclosing methods for in vitro differentiation
of embryonic
stem cells to produce various differentiated cell types including
hematopoietic cells,
muscle, cardiac muscle, nerve cells, among others. These references and in
particular the
disclosures therein relating., to methods for differentiating embryonic stem
cells are
incorporated by reference in their entirety herein.
Thus, using known methods and culture mediums, one skilled in the art may
culture the subject somatic cells and cells created using somatic cell nuclei
to obtain cells
for producing transgenic or chimeric animals.
The present invention has been described with reference to a preferred
embodiment. However, it will be readily apparent to those skilled in the art
that it is
possible to embody the invention in specific forms other than as described
above without
departing from the spirit of the invention. The embodiments described in the
examples
below are illustrative and should not be considered restrictive in any way.
The scope of
the invention is given by the appended claims, rather than the preceding
description, and
all variations and equivalents which fall within the range of the claims are
intended to be
embraced therein.
EXAMPLE 1
Effect of Confluency and Cell Age on Cell Cycle Length
Establishment of fetal cell lines. Bovine fetuses were obtained from a
slaughterhouse and the crown-rump length was measured. After washing in rinse
solution (DPBS containing antibiotic/antimycotic (Sigma) and Fungizone (Gibco)
and
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removing the head and internal organs, the remaining tissues were finely
chopped into
pieces, using scalpel blades. Tissue pieces were washed twice in rinse
solution by
allowing the pieces to settle to the bottom of 50 ml tubes and removing the
supernatant.
To the tissue pieces, 30-40 ml of 0.08% trypsin (Difco) and 0.02% EDTA (Sigma)
in PBS
(Gibco) was added, and the tissue incubated for 30 min. at 39°C., 5%
COZ. At intervals
of 30 mm., the supernatant was carefully removed and centrifuged in another
tube for 5
mm. at 300 x g. Then the tissue pieces were separated by removing the
supernatant,
adding another 30-40 ml of 0.08% trypsin and 0.02% EDTA in PBS, and incubating
the
tissue samples again for 30 min. at 39°C., at 5% CO2. The supernatant
then carefully was
removed leaving the tissue pieces in a 50 ml tube; an equal volume of alpha
MEM
(Gibco) supplemented with 10% FCS (fetal calf serum, Hyclone), glutamine
(Sigma),
mercaptoethanol (Gibco) and antibiotic/antimycotic was added to the tissue,
and the
tissue was centrifuged at 1,000 x g for 5 min. The pellet was carefully
separated by
aspirating off the supernatant. The tissue was resuspended in alpha MEM
supplemented
with the above components and seeded on 100 mm tissue culture plates (Coming)
and
incubated at 39°C., 5% COZ. The tissue pieces were again incubated with
the trypsin -
EDTA in PBS solution, the supernatant collected, and the cells seeded as
described
above. On day three of seeding, the cells were harvested, using trypsin-EDTA
solution
and counted. One million cells were selected and re-seeded in 100 mm tissue
culture
plates, and the remaining cells were frozen in alpha MEM with 10% DMSO
(Sigma).
Other adherent similarly cells can be prepared, as would be known by the
skilled artisan.
Establishment of calf and adult cell lines. Ear punches were taken (1 mm)
after
thoroughly cleaning the skin surface by clipping the hair and washing with
disinfectant.
The ear punch samples were washed three times in rinse solution, and the
cartilage
portion was separated removed out in between the outer and inner surface of
the skin.
Samples were explanted in 100 mm tissue culture plates and covered with a
glass slide in
order to prevent floating in the culture media. After making the explant, 10
ml of alpha
MEM supplemented with the components that were used in the establishment of
fetal cell
lines (above), was added and incubated at 39°C., 5% CO2. After removal
of the explants
on day 10, monolayers of cells were harvested using 0.08% trypsin and 0.02%
EDTA in
PBS solution, counted and re-seeded in 100 mm tissue culture plates.
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Population doublings and cell counts. After the initial seeding, cells were
counted when they were 90% confluent by standard trypsinization procedure
using 20
0.08% trypsin and 0.02% EDTA in PBS solution. Harvested cells were centrifuged
at
1000 x g for 5 min., and the cell pellet was resuspended in 10 ml of alpha
MEM. A
S suitable sample of cells was counted using hemocytometer. When these
cultures were
95% confluent, they were harvested, counted and the population doublings were
calculated. This procedure was repeated until these cells reach senescence.
Excess cells
obtained during the harvesting and re-seeding steps were frozen in
supplemented alpha
MEM and 10% DMSO (Sigma) and stored in liquid nitrogen.
Cell fixation, staining and flow cytometry. Cell cycle comparisons were made
between cells from different confluencies (Fig. 1). After over night fixation
of the cells in
70% ethanol, cells were washed thoroughly with chilled PBS and treated with 10
RNase
followed by incubation for 2-3hrs at 37°C. After incubation, cells were
stained with
propedium iodide (Sigma).
Isolation of dividing GI cells. 24-hours prior to "shake-off," 5.0 x 105 cells
were
plated onto 100 mm Corning tissue culture plates containing 10 ml of alpha MEM
supplemented with 10% FCS. The following day, plates were washed with PBS, and
culture medium was replaced about 1 to about 2 hours before shake-off. These
plates
were vortexed for 30-60 seconds, the medium removed and centrifuged, and the
cell
pellet resuspended in 250 ltl of culture medium.
Cells attached by a cytoplasmic bridge have just under gone cytokinesis and
are in
early G~. In the following experiments, these early G1 cells were identified
(by visual
inspection) based on this characteristic and used.
Bdru labeling of G~ doublets. G~ doublet cells were placed in Lab-Tek 4-well
culture chambers (Nunc) containing 250 ,u1 of alpha MEM supplemented with
bromodeoxyuridine (Bdru)(Boehringer Mannheim). At 0, 2, 4, and 7 hours, cells
were
fixed with 70% ethanol (in 50 mM glycine buffer, pH 2.0) for about 20 minutes.
Following fixation, cells were washed and incubated with anti-Bdru for 30 min.
at 37°C.
After 30 min., cells were washed and anti-mouse-Ig-fluorescein was added;
fixed cells
were then incubated again for an additional 30 minutes at 37°C.
Following the second
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incubation, fixed cells were washed and mounted with glycerol. The percent of
cells in
S-phase was determined using an epi-fluororescent microscope (Nikon).
Assessment of Cell-Cycle Length. GI doublets were isolated via standard
micromanipulation using a 25 ltm beveled needle. Individual doublets were
transferred
into SO A4 drops of alpha MEM supplemented with 10% FCS derived from actively
dividing cultures of fibroblasts (conditioned medium). The time of pick-off
was marked
0 hours, and every 2 hours there after isolated doublets were assessed for
cell division.
Ten microdrops per culture plate were assessed, and the proportion of cells
that divided
within 24 hours were used to calculate the mean cell-cycle length.
The results for Fig. 1 were obtained by measuring the cell cycle length of
cells at
90% confluency versus cell cycle length at 25% confluency. Cells were obtained
and
plated as described above. For the most part, cell cycle length was less for
cells grown at
25% confluency rather than at 90% confluency.
EXAMPLE 2
Effect of Time in Culture and Donor Age on Cell Growth Rate
Fig. 2 demonstrates that increased time in culture for the cells obtained as
described above, leads to a decrease in population divisions or doubling per
day
(PD/DY).
EXAMPLE 3
Length of G~ in Fibroblasts Recovered from Culture
Fibroblast cells were obtained, cultured and harvested as described in Example
1.
Fig. 3 shows the length of G~ in fibroblasts recovered from culture after pick-
off.
EXAMPLE 4
Preparing Somatic Cells for Nuclear Transfer Using Cell Shake-Off and Mitotic
Doublet Cell Selection at Low Confluency.
Cells are prepared for nuclear transplantation as described above in Example
1.
Nuclear transplantation. In vitro matured oocytes were stripped with 1.0%
hyaluronidase at 18 hpm. Oocytes were briefly washed in TL-hepes and then
stained
with Hoescht 33342 (Sigma) for 20 min. The cells were enucleated using a 18-20
~m
beveled needle. Enucleation was confirmed with UV light. The donor cell was
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transferred using a 20 ,um needle and fused in a sorbitol based medium with
one electrical
pulse of 115 mV for 20 sec (Electrocell manipulator 200, San Diego, CA) at 24
hours.
Activation. At 28 hpm, reconstructed oocytes and controls were chemically
activated using a Ca ionophore (5 mM) for 4 min. (Cal Biochem) and DMAP (200
mm)
for 3.5 hours. At 3.5 hours post activation, oocytes were briefly washed in
HCEM hepes
and transferred into culture.
In vitro culture of nuclear transfer embryos. Embryo culture was performed in
4-well tissue culture plates (Nunc), containing mouse blocked feeder layer and
O.SmI of
culture media covered with 200 ,u1 of embryo tested mineral oil (Sigma). 25-50
embryos
were placed in each well and incubated at 39°C., 5% C02. On day four,
10% FCS was
added to the culture media. On days 7 and 8, development to blastocyst was 10
recorded.
Cell numbers were described by mounting the cells with 1% Hoechst in glyceral
(Sigma).
The donor somatic cell utilized preferably is any type of adherent cell. Other
similar methods and materials may be substituted and used as would be known to
the
skilled artisan.
EXAMPLE 5
Preparing Somatic Cells for Nuclear Transfer Using Cell Shake-Off
and Mitotic Doublet Cell Selection at Low Confluency and
Extending G~ Phase Using a Cooling Step,
The GI phase of the somatic cell can be extended by placing the cells at
4°C and
performing the steps for nuclear transfer, as described above.
EXAMPLE 6
Preparing Somatic Donor Cells for Nuclear Transplantation Using Mitotic
Shake-Off and Doublet Cell Selection in Combination with Serum Starvation.
The methods and materials used above can also be utilized in combination with
agents that synchronize cells in G1, such as certain kinase inhibitors (e.g.,
KT5720,
KT5823 or KT5926). Cells can be obtained via shake-off as described above.
Cells can
then be resuspended in media containing a kinase inhibitor in any one of the
following
concentrations: KT5720 at about 11 , ,uM, KT5823 at about 1 S ,uM, KT5926 at
about 3
~.cM or K252b at about 11 ,uM. G~ phase can be increased further by placing
the cells at
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4°C if G, phase is to be further lengthened. The cells can then be
utilized as previously
described.
All references are herein incorporated by reference in their entirety.
24
